Method of purifying nucleic acids, method of extracting nucleic acids and kit for purifying nucleic acids

ABSTRACT

[Object] To provide a method of purifying nucleic acids where the operation is simple and the nucleic acids can be extracted in a short time with high efficiency. 
     [Solving Means] A method of purifying nucleic acids including the step of adsorbing substances in a sample containing nucleic acids with an ion exchange resin  10  including a positive ion exchange resin and a negative ion exchange resin. As the positive ion exchange resin, a first positive ion exchange resin and a second positive ion exchange resin having an exclusion limit molecular weight lower than that of the first positive ion exchange resin may be used.

TECHNICAL FIELD

The present technology relates to a method of purifying nucleic acids, amethod of extracting nucleic acids and a kit for purifying nucleicacids, and more particularly to a method of purifying nucleic acids byadsorbing foreign substances with an ion exchange resin.

BACKGROUND ART

A nuclear acid amplification reaction such as PCR (Polymerase ChainReaction) and LAMP (Loop-Mediated Isothermal Amplification) is appliedto a variety of fields in biotechnology. For example, in a medicalfield, a diagnosis is carried out based on base sequences of DNAs andRNAs. In an agriculture field, a DNA analysis is utilized to detect generecombinant plants.

The nuclear acid amplification reaction can efficiently amplify anddetect the nucleic acids in a minor amount of sample. However, if anextremely minor amount of the nucleic acids is included in the sample,the amount of the nucleic acids may be under the lower detection limit.Furthermore, a concentration of the nucleic acids in the sample isextremely low, the nucleic acids may not be detected because the nucleicacids to be amplified are not contained in the sample having a volumethat can be fed into a reaction site. In these cases, it is effective tofeed the nucleic acids that are extracted by purifying, concentratingetc. in advance to a reaction site.

Here, by focusing on purifying the nucleic acids, a conventional methodusing phenol/chloroform/ethanol is known. Also, a method of purifyingthe nucleic acids using a porous carrier having a nucleic acid adsorbingability is known (See Patent Document 1).

-   Patent Document 1: Japanese Patent Application Laid-open No.    2005-080555-   Patent Document 2: PCT Application Laid-open No. 2009/060847

SUMMARY OF INVENTION Problem to be Solved by the Invention

However, in the conventional method using phenol/chloroform/ethanol, theuse of toxic organic solvents is necessary and a centrifugationoperation is an extra work. In the method of purifying the nucleic acidsusing a porous carrier having a nucleic acid adsorbing ability, aplurality of steps are necessary and a simple operation is unfeasible.Accordingly, there is strong needed a simple method of purifying nucleicacids in a short time with high efficiency.

A main object of the present technology is to provide a simple method ofpurifying nucleic acids in a short time efficiently.

Means for Solving the Problem

In order to solve the above-described problem, the present technologyprovides a method of purifying nucleic acids including a step ofadsorbing substances included in a sample containing the nucleic acidswith an ion exchange resin; the ion exchange resin being a positive ionexchange resin and a negative ion exchange resin. The positive ionexchange resin allows cations of positively charged protein or a metalsalt included in the sample to be adsorbed. The negative ion exchangeresin allows negatively charged anions included in the sample other thanthe nucleic acids to be adsorbed.

As the positive ion exchange resin, a first positive ion exchange resinand a second positive ion exchange resin having an exclusion limitmolecular weight lower than that of the first positive ion exchangeresin are desirably used. Herein, the exclusion limit molecular weightrefers to a lowest molecular weight of the compound to be adsorbed bythe ion exchange resin that is difficult to enter pores of the ionexchange resin, i.e., is difficult to be adsorbed within the pores ofthe ion exchange resin. In other words, the compound having themolecular weight higher than the exclusion limit molecular weight isdifficult to be adsorbed by the ion exchange resin.

Among the positive ion exchange resins, the first positive ion exchangeresin having the higher exclusion limit molecular weight may be easy toselectively adsorb the protein. In addition, among the positive ionexchange resins, the second positive ion exchange resin having the lowerexclusion limit molecular weight may be easy to selectively adsorbmainly the cation such as the metal salt.

It is desirable that after the substances be adsorbed by the firstpositive ion exchange resin, the substances be adsorbed by the negativeion exchange resin and the second ion exchange resin. In this case, acolumn includes the first positive ion exchange resin in an upper layerand the negative ion exchange resin and the second positive ion exchangeresin in a lower layer. The sample may be flowed into the column from anupper layer side. In this way, as to the substances in the sample, theprotein in the sample is selectively adsorbed mainly by the firstpositive ion exchange resin, and the metal salt in the sample isselectively adsorbed mainly by the second positive ion exchange resinand the negative ion exchange resin, thereby improving adsorptionefficiency of the substances in the sample.

Also, the step may adsorb the substances included in the sample that isdiluted with a buffer solution by the ion exchange resin. Desirably, thebuffer solution has a pH of 4.0 to 8.0.

Desirably, the positive ion exchange resin is a strong acidic positiveion exchange resin. Desirably, counter ions of the first positive ionexchange resin are Na⁺ (sodium ions). Desirably, the counter ions of thenegative ion exchange resin are OH⁻ (hydroxide ions).

Desirably, the negative ion exchange resin is a strong basic negativeion exchange resin. Desirably, counter ions of the negative ion exchangeresin are OH⁻ (hydroxide ions).

Also, desirably, a percentage of an ion exchange capacity of thenegative ion exchange resin to the second positive ion exchange resin is50% to 150%. Herein, the ion exchange capacity refers to a total numberof exchange groups per unit amount of the ion exchange resin. Forexample, when the counter ions of the second positive ion exchange resinare H⁺, the ion exchange capacity refers to the total number of H⁺included in 1 ml of the second positive ion exchange resin. When thepercentage of the ion exchange capacity of the negative ion exchangeresin to the second positive ion exchange resin is 50% to 150%, it ispossible to more stably inhibit a variation in pH before and after theadsorption of the substances included in the sample by the ion exchangeresin.

In the method of purifying the nucleic acids, it is more desirable thata nonionic surfactant and/or a nonionic hydrophilic polymer compound beadded to the sample to adsorb the substances. By adding the nonionicsurfactant and/or the nonionic hydrophilic polymer compound to thesample, it is possible to inhibit physical adsorption of the nucleicacids to the resin.

The above-described substances are foreign substances, for example. Theforeign substances contain protein and a metal salt, for example. Theforeign substances may contain substances such as a variety of peptides,sugers, salts and low molecular anions (for example, fumaric acid,phthalic acid, humic acid and fulvic acid) that are unnecessary for theanalysis of the nucleic acids in the sample in addition to the proteinand the metal salt.

In order to solve the above-described problem, the present technologyprovides a method of purifying nucleic acids including steps of:ultrasonically treating the sample including nucleic acids, adsorbingsubstances included in the sample with a positive ion exchange resin anda negative ion exchange resin, and concentrating the nucleic acids byblocking the nucleic acids migrated by electrophoresis.

The electrophoresis may be carried out on the nucleic acids into whichan intercalator having an anionic functional group is inserted. Also,the electrophoresis may be carried out on the nucleic acids by mixingthe sample, a compound having a functional group that is reacted with acarboxyl group of the substances included in the sample by dehydrationcondensation and a condensation agent of the dehydration condensationreaction.

Furthermore, the present technology provides a kit for purifying nucleicacids including a positive ion exchange resin, a negative ion resin anda nucleic acid purifying instrument internally holding the positive ionexchange resin and the negative ion exchange resin for distributing asample including nucleic acids.

Effect of the Invention

According to the present technology, there is provided a simple methodof purifying nucleic acids in a short time efficiently.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A schematic view for illustrating steps of a method of purifyingnucleic acids according to a first embodiment of the present technology.

FIG. 2 A schematic view for conceptually illustrating a status thatforeign substances are adsorbed by a first positive ion exchange resinaccording to a first embodiment of the present technology.

FIG. 3 A schematic view for conceptually illustrating a status thatforeign substances are adsorbed by a second positive ion exchange resinand a negative ion exchange resin according to a first embodiment of thepresent technology.

FIG. 4 A schematic view for illustrating steps of a method of purifyingnucleic acids according to a second embodiment of the presenttechnology.

FIG. 5 A graph showing recovery rates of nucleic acids after anadsorption treatment by the positive ion exchange resin (Test Example1).

FIG. 6 A graph showing a result of a LAMP reaction of a sample after anadsorption treatment by the positive ion exchange resin (Test Example1).

FIG. 7 A graph showing a result of reviewing an effect of the positiveion exchange resin on recovery rates of counter ion species (Na⁺, H⁺)(Test Example 2).

FIG. 8 A graph showing a result of the LAMP reaction of the sample afteran adsorption treatment by the ion exchange resin (Examples 4 and 5).

FIG. 9 A graph showing a result of the LAMP reaction and an RT-LAMPreaction (a Tf value) of the sample after an adsorption treatment by thepositive ion exchange resin (Examples 4 and 5).

FIG. 10 A graph showing a result of the LAMP reaction and the RT-LAMPreaction (a Tt value) after an adsorption treatment by the positive ionexchange resin (Example 6).

FIG. 11 A graph showing a result of the LAMP reaction and the RT-LAMPreaction (a Tt value) after an adsorption treatment by the ion exchangeresin (Example 7).

FIG. 12 A graph showing a result of the LAMP reaction of the sample(Example 12).

FIG. 13 A graph showing a result of the RT-LAMP reaction of the sample(Example 12).

MODE(S) FOR CARRYING OUT THE INVENTION

Hereinafter, embodiments of the present disclosure will be describedwith reference to the drawings. The embodiments described below merelydepict typical embodiments of the present disclosure, and the scope ofthe present disclosure should not be construed narrower. The embodimentswill be described in the following order.

1. A kit for purifying nucleic acids and a method of purifying nucleicacids according to a first embodiment of the present technology

(1) A kit for purifying nucleic acids

(2) A method of purifying nucleic acids

2. A kit for purifying nucleic acids and a method of purifying nucleicacids according to a second embodiment of the present technology

3. A method of extracting nucleic acids

1. A Kit for Purifying Nucleic Acids and a Method of Purifying NucleicAcids According to a First Embodiment of the Present Technology

(1) A Kit for Purifying Nucleic Acids

Firstly, a kit for purifying nucleic acids used in a method of purifyingnucleic acids according to a first embodiment of the present technologywill be described referring to FIG. 1 (A). FIGS. 1 (A) to (C) areschematic views for illustrating steps of a method of purifying nucleicacids according to the first embodiment of the present technology.

In FIG. 1 (A), a kit 1 for purifying nucleic acids adsorb substances(hereinafter referred to as “foreign substances”) included in the sampleother than the nucleic acids, and purify the nucleic acids. The kit 1for purifying nucleic acids mainly includes an ion exchange resin 10 anda nucleic acid purifying instrument 3 holding the ion exchange resin 10inside and being capable of distributing the sample containing thenucleic acids. The sample used in the present technology is notespecially limited, and may be a biological sample such as a swab, anoral cavity swab, saliva, blood sera, blood plasma, peripheral bloodmonocytes, cerebrospinal fluids, feces, urine, cultured cells and biopsytissues, for example. In addition to the biological sample, the samplemay also be river water, sea water, soil or the like.

The nucleic acid purifying instrument 3 holds the ion exchange resin 10inside and distribute the sample. A shape, a material or the like of thenucleic acid purifying instrument 3 is not especially limited as long asthe nucleic acid purifying instrument 3 can hold the ion exchange resin10 inside and distribute the sample. For example, a commerciallyavailable sample tube, a chip or the like having an opening at an uppersection as shown in FIG. 1 (A). Alternatively, the nucleic acidpurifying instrument 3 may have a tube having opening at upper and lowersections. In this case, after the sample is distributed through thenucleic acid purifying instrument 3 and the sample is adsorbed by theion exchange resin 10, a separate chip or the like can be used torecover the sample flowed from the lower section.

The ion exchange resin 10 according to the present technology adsorbsthe foreign substances included in the sample. The ion exchange resin 10includes a positive ion exchange resin and a negative ion exchangeresin. For example, the foreign substances contain protein and a metalsalt. The foreign substances may contain a variety of peptides, sugers,salts, low molecular anions (for example, fumaric acid, phthalic acid,humic acid and fulvic acid) that are unnecessary for the analysis of thenucleic acids in the sample in addition to the protein and the metalsalt.

The above-described positive ion exchange resin mainly adsorbs cationicforeign substances. Desirably, the positive ion exchange resin includesa first positive ion exchange resin and a second positive ion exchangeresin having an exclusion limit molecular weight lower than that of thefirst positive exchange resin. The first positive ion exchange resin isused to mainly adsorb the protein included in the sample. The secondpositive ion exchange resin is used to mainly adsorb the positive ions(cations) such as metal ions included in the sample.

Non-limiting example of the first positive ion exchange resin can beused as long as the protein included in the sample. Desirably, the firstpositive ion exchange resin is a strong acidic positive ion exchangeresin. A fixed ion of the first positive ion exchange resin is desirablySO₃ ⁻. Non-limiting examples of the counter ions include a calcium ion(Ca²⁺), a copper ion (Cu²⁺), a zinc ion (Zn²⁺), a magnesium ion (Mg²⁺),a potassium ion (K⁺), an ammonium ion (NH₄ ⁺), a sodium ion (Na⁺), aproton (H⁺) and the like. In this regard, ion exchange strength (ionselectivity) of these ions is in the order of Ca²⁺>Cu²⁺>Zn²⁺>Mg²⁺>K⁺>NH₄⁺>Na⁺>H⁺. Accordingly, in view of the ion exchange strength, the counterions are desirably H⁺. On the other hand, when the counter ions are H⁺,a pH of the sample is likely to shift to an acidic side. In contrast,when the counter ions are Na⁺, a function to adsorb the metal saltbecomes lower than that when the counter ions are H⁺. However, it ispossible to inhibit the variation in the pH of the sample. Even if thecounter ions are Na⁺, the first positive ion exchange resin can fullyadsorb protein. Therefore, the first positive ion exchange resin isdesirably a strong acidic positive ion exchange resin (a Na⁺ type strongacidic positive ion exchange resin) having Na⁺ as the counter ions.

The second positive ion exchange resin is not especially limited as longas the cations such as the metal salt included in the sample can beadsorbed, but is desirably a strong acidic positive ion exchange resin.A fixed ion of the second positive ion exchange resin is desirably SO₃⁻. The counter ions are not especially limited. However, in view of theorder of the above-described ion exchange strength (ion selectivity),the counter ions of the second positive ion exchange rein are desirablyprotons (H⁺).

The negative ion exchange resin adsorbs the positive ions (anions)included in the sample. Non-limiting example of the negative ionexchange resin can be used as long as the anions such as the metal saltincluded in the sample. Desirably, the negative ion exchange resin is astrong basic negative ion exchange resin. The negative ion exchangeresin may be either of a I type strong basic negative ion exchange resinhaving a trimethyl ammonium group or a II type strong basic negative ionexchange resin having a dimethyl ethanol ammonium group. In the I type,the negative ion exchange strength (ion selectivity) of these ions is inthe order of HSO₄ ⁻>NO₃ ⁻>Br⁻>Cl⁻>HCO₃ ⁻>HCOO⁻>CH₃COO⁻>F⁻>OH⁻. In the IItype, the negative ion exchange strength (ion selectivity) of these ionsis in the order of HSO₄ ⁻>NO₃ ⁻>Br⁻>Cl⁻>HCO₃ ⁻>OH⁻>HCOO⁻>CH₃ ⁻COO⁻>F⁻.Among the I type and II type strong basic negative ion exchange resins,the I type strong basic negative ion exchange resin can adsorb chlorideions (Cl⁻) and the like with higher accuracy. Thus, the negative ionexchange resin is desirably the I type and includes hydroxide ions (OH⁻)as the counter ions.

The kit 1 for purifying nucleic acids may include an additive such as anon-ionic compound including a non-ionic surfactant, a non-ionichydrophilic polymer compound or the like so that the nucleic acids arenot adsorbed together with the foreign substances. Examples of thenon-ionic compound include the non-ionic surfactant such as Brij35,Tween20 and TritonX100. Also, examples of the non-ionic hydrophilicpolymer compound include polyethylene glycol, polyhydroxy ethylcellulose or the like. These illustrated non-ionic compounds may be usedalone or in combination. Furthermore, the kit 1 for purifying nucleicacids may contain a chelate additive such as EDTA.

The kit 1 for purifying nucleic acids may contain a buffer solution.Examples of a buffer agent for the buffer solution include HomoPIPES(pH: 3.9-5.1, pKa: 4.55), MES (pH: 5.5-7.0, pKa: 6.15), Bis-Tris (pH:5.7-7.3, pKa: 6.46), ADA (pH: 5.8-7.4, pKa: 6.60), PIPES (pH: 6.1-7.5,pKa: 6.80), ACES (pH: 6.0-7.5, pKa: 6.90), MOPSO (pH: 6.2-7.4, pKa:6.95), BES (pH: 6.6-8.0, pKa: 7.15), MOPS (pH: 6.5-7.9, pKa: 7.20), TES(pH: 6.8-8.2, pKa: 7.50), HEPES (pH: 6.8-8.2, pKa: 7.55), DIPSO (pH:6.9-8.1, pKa: 7.6), TAPSO (pH: 7.0-8.2, pKa: 7.7), POPSO (pH: 7.2-8.5,pKa: 7.85), HEPPSO (pH: 7.4-8.6, pKa: 7.9), EPPS (pH: 7.5-8.5, pKa:8.0), Tricine (pH: 7.8-8.8, pKa: 8.15), Bicine (pH: 7.7-9.1, pKa: 8.35),TAPS (pH: 7.7-9.1, pKa: 8.4), CHES (pH: 8.6-10.0, pKa: 9.5), CAPSO (pH:9.3-10.7, pKa: 10.0), CAPS (pH: 9.7-11.1, pKa: 10.40) or the like. ThepH range of each buffer agent cited above refers to an appropriate pHrange of the sample to which the buffer agent is added. The pKa of eachbuffer agent cited above refers to a pKa at 20° C. excluding HomoPIPES(as to HomoPIPES, the pKa is at 37° C.). The pH of the buffer solutionto which the sample is diluted is desirably 4 to 8. In this regard,HomoPIPES, MES, Bis-Tris and ADA are desirably used in the firstembodiment among the buffer agents cited above.

(2) Method of Purifying Nucleic Acids

Next, steps of a method of purifying nucleic acids according to a firstembodiment of the present technology will be described referring toFIGS. 1 (A) to (C).

Before describing the method of purifying nucleic acids according to thefirst embodiment of the present technology, a method of purifyingnucleic acids in the related art of the present technology will bedescribed. In the method of purifying nucleic acids in the related art,foreign substances in the sample are adsorbed using zeolite (see PatentDocument 2, for example).

By the method of purifying nucleic acids in the related art, onlycations among anions and cations included in the sample such as thebiological sample can be removed. Thus, in the method of purifyingnucleic acids in the related art, when the sample is purified, protonswill be discharged, thereby decreasing the pH of the solution containingthe sample (more shifting to the acidic side). In this regard, after thesample is purified, the nucleic acid amplification reaction may becarried out at the pH of 7 to 9. It is necessary to set the pH of thesolution containing the sample being contacted with zeolite higher inadvance (to set it at an alkali side). In this way, the method ofpurifying nucleic acids in the related art may be a cumbersomeoperation. Also, when the sample includes RNAs, the RNAs may bedecomposed.

In the method of purifying nucleic acids in the related art, after thesample is diluted with a mixed reagent of an alkaline compound and asurfactant (for example, SDS), the sample is heated at high temperature.Also in this regard, the method of purifying nucleic acids in therelated art may be a cumbersome operation. Also, when the sampleincludes RNAs, the RNAs may be decomposed.

In addition, in the method of purifying nucleic acids in the relatedart, after the sample is purified, anions may remain in the sample.Further, an anionic surfactant may be included in the sample. Thus, whenthe nucleic acids are electrophoresed after purification of the sample,the sample may have a high electrolyte concentration and easily generateheat. Also in this regard, when the sample includes RNAs, the RNAs maybe decomposed. In addition, a convention flow of the sample is inducedby the heat generation, thereby decreasing efficiency of electrophoresiscondensation of the nucleic acids.

In contrast, the method of purifying nucleic acids according to thepresent technology described below in detail has been found by thepresent inventors through their intensive studies, needs no cumbersomeoperation, very simplifies the operation, and is a simple method capableof purifying nucleic acids in a short time with high efficiency.

In the method of purifying nucleic acids according to the firstembodiment of the present technology, a sample A containing nucleicacids housed in a container 5 and a buffer solution for diluting thesample A are filled into a pipette tip 51 attached to a pipette 50 (seeFIG. 1 (A)). Next, the sample A and the buffer solution for diluting thesample A filled into the pipette 50 are injected into a nucleic acidpurifying instrument 3 (see FIG. 1 (B)). At this time, as the nucleicacid purifying instrument 3 holds the ion exchange resin 10 inside, theforeign substances included in the sample A are adsorbed by the ionexchange resin 10.

Finally, the sample A purified by adsorbing the foreign substances withthe ion exchange resin 10 passes through the ion exchange resin 10 andis pooled at a bottom of the nucleic acid purifying instrument 3 (seeFIG. 1 (C)).

After the steps shown in FIGS. 1 (A) to (C), the nuclear acidamplification reaction can be carried out on the sample A. For example,the nuclear acid amplification reaction can be carried out by adding thesample A to a solidified nuclear acid amplification reagent. In thefirst embodiment, the sample may be demineralized by the ion exchangeresin 10 to change double helical DNAs into single stranded DNAs. Thus,the DNAs may be modified as appropriate before the nuclear acidamplification reaction.

Here, referring to FIGS. 2 and 3, a status that the foreign substancesincluded in the sample A are adsorbed by the ion exchange resin 10 ((I)in FIG. 1 (B)) will be described in detail. FIG. 2 is a schematic viewfor conceptually illustrating the status that the foreign substances areadsorbed especially by the first positive ion exchange resin shown inFIG. 1 (B). FIG. 3 is a schematic view for conceptually illustrating thestatus that the foreign substances are adsorbed especially by the secondpositive ion exchange resin and the negative ion exchange resin shown inFIG. 1 (B).

Referring to FIG. 2, the status that the foreign substances included inthe sample A are adsorbed by a first positive ion exchange resin 20included in the ion exchange resin 10. In FIG. 2, nucleic acids B areincluded in the sample A and protein C is one of the foreign substancesincluded in the sample A. Here, the first positive ion exchange resin 20will be the strong acidic positive ion exchange resin having sodium ions(Na⁺) as the counter ions.

The first positive ion exchange resin 20 includes anions 21 such as SO₃⁻ and the counter ions Na⁺ 22. In addition, areas 23 for adsorbing theprotein C is formed on the first positive ion exchange resin 20.

As shown in FIG. 2, as the protein C included in the sample A flowedinto the nucleic acid purifying instrument 3 is positively charged, theprotein C is adsorbed by the first positive ion exchange resin 20 havingthe anions 21 on the surface. For example, the protein C is adsorbed atthe areas 23. On the other hand, as the nucleic acids B are negativelycharged, the nucleic acids B are not adsorbed by the first positive ionexchange resin 20, and are induced by and flows through the buffersolution or the like included in the sample A. In addition, as thecounter ions of the first positive exchange resin 20 are Na⁺ 22, thefirst positive exchange resin 20 less adsorb (demineralize) the metalsalt but can selectively adsorb the protein C, as compared with the casewhere the counter ions are H⁺.

In this case, the buffer solution for diluting the sample A hasdesirably the pH of 4 to 8. With the buffer solution for diluting thesample A having the pH of 4 to 8 (acidic), only the foreign substances(mainly, the protein C) can be positively charged while the nucleicacids B are negatively charged. In this way, only the protein C can bestably adsorbed by the ion exchange resin 20 and the nucleic acids B canbe purified with high efficiency. In addition, as the first positive ionexchange resin 20 is the strong acidic positive ion exchange resin, thefirst positive ion exchange resin 20 can be negatively charged under awide range of the pH conditions (for example, pH of 3 to 13). Asdescribed above, even if the sample is acidic, the first positive ionexchange resin 20 can stably adsorb the protein C. Furthermore, with thesample A having the pH of 4 to 8 (acidic), activity of RNases can besuppressed, thereby favorably purifying the RNA.

The exclusion limit molecular weight of the first positive ion exchangeresin 20 is desirably such that the protein C included in the sample Ais brought into the areas 23. More specifically, the exclusion limitmolecular weight of the first positive ion exchange resin 20 isdesirably 5000 or more. When the exclusion limit molecular weight of thefirst positive ion exchange resin 20 is 5000 or more, the protein C iseasily and particularly selectively adsorbed and removed from theforeign substances.

A mean volume particle diameter of the first positive ion exchange resin20 is desirably 1 μm to 3000 μm. When the mean volume particle diameterof the first positive ion exchange resin 20 is 1 μm to 3000 μm, theprotein C is easily and particularly selectively adsorbed and removedfrom the foreign substances.

A specific gravity of particles themselves in the first positive ionexchange resin 20 is desirably 0.5 to 2.5. When the specific gravity ofthe first positive ion exchange resin 20 is 0.5 to 2.5, the protein C iseasily and particularly selectively adsorbed and removed from theforeign substances. More desirably, the specific gravity of theparticles themselves is 1.0 to 2.5. When the specific gravity is 1.0 to2.5, the particles are easily settled within the solution, whereby it ispossible to easily remove the particles from the solution.

Next, FIG. 3 illustrates the status that the foreign substances includedin the sample A are adsorbed by a negative ion exchange resin 30 and asecond positive ion exchange resin 40 included in the ion exchange resin10.

In FIG. 3, each designated symbol D1, D2 or D3 is the cation or theanion included in the metal salt that is an example of the foreignsubstances included in the sample A. Although it is not especiallylimited in the first embodiment, D1 denotes the anion such as a chlorideion, D2 denotes the cation such as a sodium ion, and D3 denotes thecation such as a magnesium ion.

Referring to FIG. 3, the status that the negative ion exchange resin 30adsorbs the foreign substances (mainly the anions such as the chlorideion) will be described. In the first embodiment, the negative ionexchange resin 30 is the I type strong basic negative ion exchange resinhaving hydroxide ions (OH⁻) as the counter ions.

The negative ion exchange resin 30 has cations 31 such as CH₂N(CH₃)₃ ⁺and the counter ions such as OH⁻ 32. In addition, areas 33 for adsorbingthe anions such as the chloride ion D1 are formed in the negative ionexchange resin 30.

As shown in FIG. 3, the chloride ion D1 included in the sample A flowedinto the nucleic acid purifying instrument 3 is negatively charged, andis adsorbed by the negative ion exchange resin 30 having the cations 31on the surface. For example, the chloride ion D1 is adsorbed onto theareas 33. Although the nucleic acid B is also negatively charged, thenucleic acid B has a volume larger than the anion such as the chlorideion D1 and is therefore less adsorbed by the negative ion exchange resin30. In this regard, the exclusion limit molecular weight of the negativeion exchange resin 30 is desirably 100 to 2000. When the exclusion limitmolecular weight of the negative ion exchange resin 30 is 100 to 2000,the adsorption of the nucleic acid B is inhibited with higher accuracyand the cations such as the chloride ion D1 are selectively adsorbed andeasily removed.

A mean volume particle diameter of the negative ion exchange resin 30 isdesirably 1 μm to 3000 μm. When the mean volume particle diameter of thefirst positive ion exchange resin 20 is 1 μm to 3000 μm, the cationssuch as the chloride ion D1 are selectively adsorbed and easily removed.

A specific gravity of the negative ion exchange resin 30 is desirably0.5 to 2.5. When the specific gravity of the negative ion exchange resin30 is 0.5 to 2.5, the cations such as the chloride ion D1 areselectively adsorbed and easily removed with higher accuracy. Moredesirably, the specific gravity of the particles themselves is 1.0 to2.5. When the specific gravity is 1.0 to 2.5, the particles are easilysettled within the solution, whereby it is possible to easily remove theparticles from the solution.

Next, FIG. 3 illustrates the status that the foreign substances (mainlythe cations of the sodium ion D2 and the magnesium ion D3) are adsorbedby the second positive ion exchange resin 40. In the first embodiment,the second positive ion exchange rein 40 is the strong acidic positiveion exchange resin having H⁺ ions (protons) as the counter ions.

The second positive ion exchange rein 40 has anions 41 such as SO₃ ⁻ andH⁺ 42. In addition, areas 43 for adsorbing the cations such as thesodium ion D2 and the magnesium ion D3 are formed in the second positiveion exchange resin 40.

As shown in FIG. 3, the sodium ion D2 and the magnesium ion D3 includedin the sample A flowed into the nucleic acid purifying instrument 3 arepositively charged, and are adsorbed by the second positive ion exchangeresin 40 having the anions 41 on the surface. For example, the sodiumion D2 and the magnesium ion D3 are adsorbed onto the areas 43. On theother hand, the nucleic acids B are negatively charged, are thereforenot adsorbed by the second positive ion exchange resin 40, and areinduced by and flow through the buffer solution included in the sampleA.

Here, the second positive ion exchange resin 40 has the exclusion limitmolecular weight lower than that of the first positive ion exchangeresin 20. Accordingly, the second positive ion exchange resin 40 has anincreased surface area per unit area, and is easily demineralized ascompared to the first positive ion exchange resin 20. When the secondpositive ion exchange resin 40 has protons as the counter ions, thesecond positive ion exchange resin 40 is easily demineralized ascompared to the first positive ion exchange resin 20. The exclusionlimit molecular weight of the second positive ion exchange resin 40 isdesirably 100 to 2000. When the exclusion limit molecular weight of thesecond positive ion exchange resin 40 is 100 to 2000, the adsorption ofthe cations such as the sodium ion D2 and the magnesium ion D3 can beadsorbed with higher accuracy.

A mean volume particle diameter of the second positive ion exchangeresin 40 is desirably 1 μm to 3000 μm. When the mean volume particlediameter of the second positive ion exchange resin 40 is 1 μm to 3000μm, the cations such as the sodium ion D2 and the magnesium ion D3 canbe adsorbed with higher accuracy. The mean volume particle diameter ofthe second positive ion exchange resin 40 is more desirably 1 μm to 2000μm. When the mean volume particle diameter of the second positive ionexchange resin 40 is 1 μm to 2000 μm, it is possible to decrease apressure when the solution flows through the ion exchange resin.

A specific gravity of the second positive ion exchange resin 40 isdesirably 0.5 to 2.5. When the specific gravity of the second positiveion exchange resin 40 is 0.5 to 2.5, the cations such as the sodium ionD2 and the magnesium ion D3 can be adsorbed with higher accuracy. Moredesirably, the specific gravity of the particles themselves is 1.0 to2.5. When the specific gravity is 1.0 to 2.5, the particles are easilysettled within the solution, whereby it is possible to easily remove theparticles from the solution.

Also, desirably, the percentage of the ion exchange capacity of thenegative ion exchange resin 30 to the second positive ion exchange resin40 is 50% to 150%. When the percentage of the ion exchange capacity is50% to 150%, the sample can be demineralized while the variation in thepH is more stably inhibited.

Also, desirably, a percent amount of the negative ion exchange resin 30to the second positive ion exchange resin 40 is 50% to 150%. When thepercent amount is 50% to 150%, the sample can be demineralized while thevariation in the pH is more stably inhibited.

Although not shown in FIGS. 2 and 3, the non-ionic surfactant such asBrij35, Tween20 and TritonX100 is added to the sample as appropriate inorder to inhibit the nucleic acids B from adsorbing by the ion exchangeresin. Also, the non-ionic hydrophilic polymer compound such aspolyethylene glycol and polyhydroxy ethyl cellulose is desirably addedto the sample A as appropriate.

According to the method of purifying nucleic acids of the firstembodiment of the present technology as described above, the ionexchange resin 10 including the positive ion exchange resin and thenegative ion exchange resin is used as an adsorbing carrier, therebyadsorbing the foreign substances included in the sample. For example,when the sample if a blood sample, the step of adsorbing can be directlycarried out with no complex pretreatment steps. Thus, the method ofpurifying nucleic acids according to the first embodiment can extractthe nucleic acids by a very simple operation in a short time with highefficiency. Specifically, only a few seconds is necessary to conduct allsteps in the method of purifying nucleic acids. In this way, the nucleicacids can be purified in such a short time.

As the method of purifying nucleic acids according to the firstembodiment includes no cleaning steps dissimilar to a silica solid-phaseextraction, for example, only a minor amount of the sample is necessaryfor the operation and the apparatus can also be downsized. Also, themethod of purifying nucleic acids according to the first embodiment canadsorb the foreign substances such that the sample is held under theacidic condition (desirably pH of 4 to 8). In other words, the method ofpurifying nucleic acids according to the first embodiment does not needthe sample to be changed to alkali or heated. Accordingly, when thesample contains RNAs, the sample can be purified while the RNAs areinhibited from decomposing. In addition, in the method of purifyingnucleic acids according to the first embodiment, when an ultrasonicdisintegration is carried out at the acidic area, the function of RNasecan be suppressed.

When the nuclear acid amplification reaction is carried out by using thesolidified nuclear acid amplification reagent, the sample is not dilutedand the foreign substances obviously inhibit the nuclear acidamplification reaction. However, as the sample purified by the method ofpurifying nucleic acids according to the first embodiment includes noforeign substances, the nuclear acid amplification reaction can becarried out by adding the nucleic acids directly to the solidifiednuclear acid amplification reagent.

According to the method of purifying nucleic acids of the firstembodiment of the present technology, the first positive ion exchangeresin and the second positive ion exchange resin having the exclusionlimit molecular weight lower than that of the first positive ionexchange resin can be used as the positive ion exchange resin.Accordingly, when the foreign substances in the sample contain the metalsalt and the protein, both of them can be efficiently removed.

To adsorb and remove the foreign substances in the sample by the methodof purifying nucleic acids according to the first embodiment, it is moreeffective that the first positive ion exchange resin and the secondpositive ion exchange resin each is the strong acidic positive ionexchange resin. The foreign substances are removed more efficiently whenthe counter ions of the first positive ion exchange resin are Na⁺, orthe counter ions of the second positive ion exchange resin are H⁺.

In the method of purifying nucleic acids according to the firstembodiment, the foreign substances are more efficiently removed when thenegative ion exchange resin is the strong basic positive ion exchangeresin. When the counter ions of the negative ion exchange resin are OH⁻,the foreign substances are more efficiently removed.

According to the method of purifying nucleic acids according to thefirst embodiment, the percentage of the ion exchange capacity of thenegative ion exchange resin to the second positive ion exchange resin is50% to 150%, whereby the variation in the pH in the sample can beinhibited. As described above, when the sample contains RNAs, the samplecan be purified while the RNAs are inhibited from decomposing.

In the method of purifying nucleic acids according to the firstembodiment, the nonionic compound such as the non-ionic surfactant andthe non-ionic hydrophilic polymer compound can be used. In this case,the nucleic acids are inhibited from adsorbing to a matrix of the ionexchange resin and the foreign substances can be efficiently removed.

2. A Kit for Purifying Nucleic Acids and a Method of Purifying NucleicAcids According to a Second Embodiment of the Present Technology

FIGS. 4 (A) to (C) are schematic views for illustrating steps of amethod of purifying nucleic acids according to the second embodiment ofthe present technology.

In FIG. 4 (A), a kit 101 for purifying nucleic acids includes the ionexchange resin 10 and the nucleic acid purifying instrument 3 holdingthe ion exchange resin 10 inside and being capable of distributing thesample containing the nucleic acids. The ion exchange resin 10 includesthe positive ion exchange resin and the negative ion exchange resin. Inthe kit 101 for purifying nucleic acids according to the secondembodiment, the positive ion exchange resin includes the first positiveion exchange resin 20 and the second positive ion exchange resin 40.

The kit for purifying nucleic acids and the method of purifying nucleicacids according to the second embodiment is mainly different from thekit for purifying nucleic acids and the method of purifying nucleicacids according to the first embodiment in that the nucleic acids arepurified while the first positive ion exchange resin 20 is housed in anupper layer (at a side where the sample is injected) of the negative ionexchange resin 30 and the second positive ion exchange resin 40. Forthis purpose, the position of the first positive ion exchange resin 20disposed at the upper layer of the negative ion exchange resin 30 andthe second positive ion exchange resin 40 will be mainly described inthe second embodiment.

In the method of purifying nucleic acids according to the secondembodiment, the first positive ion exchange resin 20 is housed in anupper layer side of the negative ion exchange resin 30 and the secondpositive ion exchange resin 40 within the nucleic acid purifyinginstrument 3. Thus, the sample A injected from the upper layer side isfirstly contacted with the first positive ion exchange resin 20 (seeFIG. 4 (B)).

Next, the sample A where a part of the foreign substances are removed bythe first positive ion exchange resin 20 is contacted with the negativeion exchange resin 30 and the second positive ion exchange resin 40. Thestatus that the protein contained in the foreign substances in thesample A is adsorbed by the first positive ion exchange resin 20 (area(II) in FIG. 4 (B)) can be explained similar to the status that theprotein is adsorbed by the first positive ion exchange resin 20referring to FIG. 2. Therefore, the detailed description is omitted. Thestatus that the metal salt contained in the foreign substances in thesample A is adsorbed by the negative ion exchange resin 30 and thesecond positive ion exchange resin 40 (area (III) in FIG. 4 (B)) can beexplained similar to the status that the metal salt is adsorbed by thenegative ion exchange resin 30 and the second positive ion exchangeresin 40 referring to FIG. 3. Therefore, the detailed description isomitted. Either the negative ion exchange resin 30 or the secondpositive ion exchange resin 40 may be housed as the upper layer thereof,or may be mixed and housed as a lower layer of the first positive ionexchange resin 20.

Finally, after the foreign substances are adsorbed by the ion exchangeresin 10, the purified sample A is pooled at the lower layer (see FIG. 4(C)).

According to the method of purifying nucleic acids according to thesecond embodiment of the present technology, the foreign substances inthe sample A are adsorbed by the first positive ion exchange resin 20and then are further adsorbed by the negative ion exchange resin 30 andthe second positive ion exchange resin 40. Thus, according to the methodof purifying nucleic acids according to the second embodiment of thepresent technology, after the protein having a higher volume is removed,the metal salt having a lower volume can be removed, thereby efficientlyremoving the foreign substances.

3. A Method of Extracting Nucleic Acids

Next, a method of extracting nucleic acids including the method ofpurifying nucleic acids according to the respective embodiments will bedescribed. The method of extracting nucleic acids according to thepresent technology includes the step of ultrasonically treating a sampleincluding nucleic acids, adsorbing substances included in the samplewith a positive ion exchange resin and a negative ion exchange resin,and concentrating the nucleic acids by blocking the nucleic acidsmigrated by electrophoresis as pretreatments for the nuclear acidamplification reaction.

The above-described steps are not especially limited. For example, allsteps can be carried out within the same cell. When the respective stepsare carried out within the same cell, an ultrasonic generator for anultrasonic process can be disposed in the cell. In addition, the cellcan house the positive ion exchange resin and the negative ion exchangeresin. As to the positive ion exchange resin and the negative ionexchange resin, the ion exchange resin 10 (the first positive ionexchange resin 20, the negative ion exchange resin 30 and the secondpositive ion exchange resin 40) that are used in the method of purifyingnucleic acids according to the respective embodiments of the presenttechnology can be used. Also, a negative electrode and a positiveelectrode for electrophoresis are disposed in the cell, and a blockerfor blocking the nucleic acids electrophoresed (e.g., a dialysismembrane, a polymer gel or the like) can be disposed in the cell.

Upon the electrophoresis of the nucleic acids, the intercalator havingthe anionic functional group can be inserted into the nucleic acids.Examples of the intercalator include a compound having a sulfofunctional group. Specific examples include9,10-anthraquinone-2,6-disulfonic acid, anthraquinone-1-sodiumsulfonate, anthraquinone-2,7-disodium sulfonate,anthraquinone-1,5-disodium sulfonate, anthraquinone-2-sodium sulfonateand the like. In this way, when the intercalator having the anionicfunctional group is inserted into the nucleic acids, an isoelectricpoint of the nucleic acids can be adjusted in the electrophoresis of thenucleic acids. In other words, the nucleic acids can be concentrated andpurified, while an electrophoretic velocity of the nucleic acids iscontrolled.

Furthermore, carboxyl groups of the protein that is one of the foreignsubstances included in the sample may be dehydrated and condensed with acompound such as N-hydroxy succin imide (NHS), ethanol amine, ethylenediamine or the like. In the dehydration and condensation reaction, acarbodiimide-based compound can be used as a condensation agent.Specific examples of the carbodiimide-based compound include1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC),4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMT-MM), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC)or the like. Thus, even if the protein is present in the sample upon theelectrophoresis of the nucleic acids, a difference between theisoelectric point of the nucleic acids that are dehydrated and condensedwith the carboxyl groups in the protein and that of the protein isenlarged, thereby separating the nucleic acid from the protein with highaccuracy.

By carrying out the above-described respective steps, the nucleic acidsconcentrated can be recovered very simply and with high efficiency. Inparticularly, when the respective steps are carried out within the samecell, an infectious risk to an operator can be reduced when aninfectious specimen is handled as the sample, because a transfer of thesample to the other apparatuses is not necessary.

The present technology may have the following configurations.

(1) A method of purifying nucleic acids, including the step of:

adsorbing substances in a sample containing nucleic acids with an ionexchange resin including a positive ion exchange resin and a negativeion exchange resin.

(2) The method of purifying nucleic acids according to (1) above, inwhich

the positive ion exchange resin includes a first positive ion exchangeresin and a second positive ion exchange resin having an exclusion limitmolecular weight lower than that of the first positive ion exchangeresin.

(3) The method of purifying nucleic acids according to (2) above, inwhich

the substances are adsorbed by the first positive ion exchange resin andthen are further adsorbed by the negative ion exchange resin and thesecond positive ion exchange resin.

(4) The method of purifying nucleic acids according to (2) or (3) above,in which

the sample is flowed into a column including the first positive ionexchange resin in an upper layer and the negative ion exchange resin andthe second positive ion exchange resin in a lower layer from an upperlayer side.

(5) The method of purifying nucleic acids according to any one of (1) to(4) above, in which

the step is for adsorbing the substances included in the sample that isdiluted with a buffer solution by the ion exchange resin, and the buffersolution has a pH of 4.0 to 8.0.

(6) The method of purifying nucleic acids according to any one of (1) to(5) above, in which

the positive ion exchange resin is a strong acidic positive ion exchangeresin.

(7) The method of purifying nucleic acids according to any one of (2) to(6) above, in which

a counter ion of the first positive ion exchange resin is Na⁺.

(8) The method of purifying nucleic acids according to any one of (2) to(7) above, in which

a counter ion of the first positive ion exchange resin is H⁺.

(9) The method of purifying nucleic acids according to any one of (1) to(8) above, in which

the negative ion exchange resin is a strong basic negative ion exchangeresin.

(10) The method of purifying nucleic acids according to any one of (1)to (9) above, in which

a counter ions of the negative ion exchange resin is OH⁻.

(11) The method of purifying nucleic acids according to any one of (2)to (10) above, in which

a percentage of an ion exchange capacity of the negative ion exchangeresin to the second positive ion exchange resin is 50% to 150%.

(12) The method of purifying nucleic acids according to any one of (1)to (11) above, in which

a nonionic surfactant and/or a nonionic hydrophilic polymer compound isused to adsorb the substances.

(13) The method of purifying nucleic acids according to any one of (1)to (12) above, in which

the substances are foreign substances containing at least a protein anda metal salt.

(14) A method of extracting nucleic acids, including the steps of:

ultrasonically treating the sample including nucleic acids,

adsorbing substances included in the sample with a positive ion exchangeresin and a negative ion exchange resin, and

concentrating the nucleic acids by blocking the nucleic acids migratedby electrophoresis.

(15) The method of extracting nucleic acids according to (14) above, inwhich

the electrophoresis is carried out on the nucleic acids into which anintercalator having an anionic functional group is inserted.

(16) The method of extracting nucleic acids according to (14) or (15)above, in which

the electrophoresis is carried out on the nucleic acids by mixing thesample, a compound having a functional group that is reacted with acarboxyl group of the substances included in the sample by dehydrationcondensation, and a condensation agent of the dehydration condensationreaction.

(17) A kit for purifying nucleic acids including a positive ion exchangeresin; a negative ion resin; and a nucleic acid purifying instrumentinternally holding the positive ion exchange resin and the negative ionexchange resin for distributing a sample including nucleic acids.

EXAMPLES 1. Effect of Adsorption Treatment by Strong Acidic Positive IonExchange Resin on Purifying Ability of Nucleic Acids Test Example 1

100 mg of a strong acidic positive ion exchange resin (Nuvia S Na⁺ type(manufactured by Bio-Rad Laboratories, Inc.)) was weighed and chargedinto a spin filter column (Ultrafree-MC, 0.45 μm, manufactured byMillipore). Next, 50 mM MES buffer (pH of 5) (manufactured by DojindoMolecular Technologies, Inc.) was prepared. To the buffer, 0.5% by massof BSA (manufactured by Wako Pure Chemical Industries, Ltd.) was addedand then 5 μM of Cy3-modified 20 mer oligoDNAs (manufactured by SigmaAldrich Co.) was added. After a blended liquid of the protein and thenucleic acids was sufficiently agitated at normal temperature, 200 μL ofthe blended liquid was dropped into a spin column filled with the strongacidic positive ion exchange resin and was sufficiently agitated.Thereafter, the blended liquid was centrifuged and spun down at 12000 Gfor 2 minutes. The spun-down liquid was measured for absorbance usingNanoDrop D-1000 (manufactured by Thermo Fisher Scientific Inc.). From adifference between the absorbances before and after the treatment by thestrong acidic positive ion exchange resin, a purifying ability ofnucleic acids was evaluated. The absorbance of the BSA was evaluated ina protein A280 mode whereas the absorbance of the nucleic acids wasevaluated as the absorbance of Cy3 in a micro array mode. Furthermore,in order to close to a biological environment, to the protein-nucleicacid blended liquid, 0.09% by mass (at a final concentration) of NaCl(manufactured by Wako Pure Chemical Industries, Ltd.) was added.Similarly, the treatment was carried out by the strong acidic positiveion exchange resin.

Results of the evaluation are shown in FIG. 5. As shown in FIG. 5, fromthe concentration of the Cy3 oligoDNAs and the BSA before and after thetreatment by the strong acidic positive ion exchange resin, the recoveryrate of the nucleic acids including NaCl was about 100%, and therecovery rate of the nucleic acids including no NaCl was about 72%. Onthe other hand, the recovery rate of the BSA including NaCl was about20%, and the recovery rate of the BSA including no NaCl was about 14%.From these facts, it revealed that, by carrying out the treatment by thestrong acidic positive ion exchange resin, a DNA presence ratio can beincreased in a blended system of the protein and the nucleic acids.

FIG. 6 shows a result of a LAMP reaction of a sample after an adsorptiontreatment of foreign substances by the positive ion exchange resin and asample without the adsorption treatment. A LAMP reaction liquid wasprepared by blending the sample, an enzyme, a fluorescent pigment, anucleic acid monomer, a buffer, a primer set for a target nucleic acidchain amplification and a probe for real-time measurements. Eachconcentration was determined by a Loopamp DNA amplification kit(manufactured by Eiken Chemical Co., Ltd.) and a Loopamp RNAamplification kit (manufactured by Eiken Chemical Co., Ltd.). Thereaction temperature was at 63° C. The LAMP reaction was measured bymaking use of a thermal cycler Chromo4 (manufactured by Bio Rad, US)capable of carrying out real-time measurements. 1 cycle was set to 1min. The used probe was a QP probe which was a quenching probe so thatthe amplification of the nucleic acids and the decrease of thefluorescence intensity can be observed. For more information on the QPprobe which is J-bio21, refer to a Japanese website,http://www.j-bio21.co.jp/tech/qpmethod.htm dated Jul. 19, 2011 with atitle of “QP Method.” As shown in FIG. 6, the nuclear acid amplificationreaction was detected on the sample after the adsorption treatment ofthe foreign substances by the ion exchange resin. The LAMP reaction asdescribed later was carried out as described above.

2. Review about Counter Ion of Positive Ion Exchange Resin Test Sample 2

100 mg of a positive ion exchange resin (Nuvia S Na⁺ type (manufacturedby Bio-Rad Laboratories, Inc.)) was weighed and charged into a spinfilter column (Ultrafree-MC, 0.45 μm). The above-described positive ionexchange resin (Nuvia S Na⁺ type) was flowed through a 1M HCl solutionand was cleaned with pure water to be neutral to prepare a positive ionexchange resin (Nuvia S H⁺ type). 100 mg of the positive ion exchangeresin (Nuvia S H⁺ type) was weighed and charged into the spin filtercolumn (Ultrafree-MC, 0.45 μm). Next, 50 mM MES buffer (pH of 5) wasprepared. To the buffer, 0.5% by mass of BSA was added and then 4 μM ofCy3-modified 20 mer oligoDNAs was added. In addition, 0.09% by mass ofNaCl was added. After each sample was sufficiently agitated at normaltemperature, 200 μL of each sample was dropped into a spin column filledwith a Na⁺ type strong acidic positive ion exchange resin or a H⁺ typestrong acidic positive ion exchange resin and was sufficiently agitated.Thereafter, the blended liquid was centrifuged and spun down at 12000 Gfor 2 minutes. The purifying ability of nucleic acids by each strongacidic positive ion exchange resin was evaluated similar to the textexample 1. The concentration of Na⁺ was measured using Horiba CardySodium Compact Ion Meter (C-122) (manufactured by HORIBA Ltd.) The pHwas measured using a pocket pH meter S2K712 (manufactured by ISFETCOMCo., Ltd.). Results of the evaluation are shown in FIG. 7. A summary ofthe evaluation results are shown in Table 1.

TABLE 1 Before adsorption Na⁺ type H⁺ type Recovery rate 100 100 26 ofnucleic acids (%) Recovery rate 100 20 18 of BSA (%) Recovery rate 1.05.2 1.5 of nucleic acids/recovery rate of BSA Na⁺ 450 450 46concentration (ppm) pH 4.9 5.2 2.2

It revealed that the recovery rate of the nucleic acids and thepurifying rate were higher when the Na⁺ type strong acidic positive ionexchange resin was used than those when the H⁺ type strong acidicpositive ion exchange resin. On the other hand, the concentration of washigher and demineralization less occurred when the Na⁺ type strongacidic positive ion exchange resin was used as compared with when the H⁺type strong acidic positive ion exchange resin was used.

It revealed that the pH of the sample after the adsorption treatment waslower than the pH of the sample before the adsorption treatment when theH⁺ type strong acidic positive ion exchange resin was used as comparedwith when the Na⁺ type strong acidic positive ion exchange resin wasused. This may because negative charges of the nucleic acids wereweakened, the nucleic acids were easily held non-specifically on the H⁺of the strong acidic positive ion exchange resin and the recovery rateof the nucleic acid was decreased when the H⁺ type strong acidicpositive ion exchange resin was used as compared with when the Na⁺ typestrong acidic positive ion exchange resin was used.

3. Review about Salt Concentration Test Example 3

This Test Example reviews an effect of a salt concentration in a samplewhen the positive ion exchange resin adsorbs the foreign substance inthe sample.

100 mg of a positive ion exchange resin (Nuvia S Na⁺ type (manufacturedby Bio-Rad Laboratories, Inc.)) was weighed and charged into a spinfilter column (Ultrafree-MC, 0.45 μm). Next, 50 mM MES buffer (pH of 5)was prepared. To the buffer, 0.5% by mass of BSA was added, then 4 μM ofCy3-modified 20 mer oligoDNAs was added, and further 3.3, 0.9 or 0.33mg/mL of NaCl was added to prepare three samples. Another sample wasprepared under the condition similar to the above-described threesamples except that no NaCl was added. After each samples wassufficiently agitated at normal temperature, 200 μL of each sample wasdropped into a spin column filled with a Na+ type strong acidic positiveion exchange resin or a H⁺ type strong acidic positive ion exchangeresin and was sufficiently agitated. Thereafter, the blended liquid wascentrifuged and spun down at 12000 G for 2 minutes. The purifyingability of nucleic acids by each strong acidic positive ion exchangeresin was evaluated similar to the text examples 1 and 2. A summary ofevaluation results are shown in Table 2.

TABLE 2 NaCl concentration (mg/mL) 3.3 0.9 0.33 0 Recovery rate of 99100 100 79 nucleic acids (%) Recovery rate of 20 20 20 17 BSA (%)Recovery rate of 5.0 5.2 4.9 4.6 nucleic acids (%)/recovery rate of BSA(%)

It revealed that the recovery rate of the nucleic acids was higher whenthe NaCl concentration was 0.33, 0.9 or 3.3 mg/ml as compared with whenthe concentration was 0 mg/mL. The recovery rate of the nucleic acidswas 100% when the NaCl concentration was 0.33, 0.9 or 3.3 mg/ml. Basedon the results, when the sample including the salt such as blood is usedto carry out the nucleic acid purification, addition of the salt to thesample upon purification may not be necessary. On the other hand, whenthe sample including no salt such as bacteria is used to carry out thenucleic acid purification, addition of the salt to the sample uponpurification may be desirable.

4. Effect of Ion Exchange Resin Species on Demineralization

Hereinbelow, an effect of ion exchange resin species on demineralizationwas reviewed in Examples 1 to 3 and Comparative Examples 1 and 2.

Example 1

100 mg of a strong acidic positive ion exchange resin (Nuvia S (Na⁺type) (manufactured by Bio-Rad Laboratories, Inc.)), 50 mg of a strongacidic positive ion exchange resin (AG1-X8 (H⁺ type) (manufactured byBio-Rad Laboratories, Inc.)) and 50 mg of a strong basic negative ionexchange resin (AG50W-X8 (OH⁻ type) (manufactured by Bio-RadLaboratories, Inc.)) were weighed and charged into a spin filter column(Ultrafree-MC, 0.45 μm). Before the adsorption treatment, theconcentration of Na⁺ was 0.45 mg/mL and the pH was 4.9. Next, 50 mM MESbuffer (pH of 5) was prepared. To the buffer, bovine whole blood wasadded to dilute to 1/10 times, thereby preparing a sample. After thesample was sufficiently agitated at normal temperature, 200 μL of thesample was dropped into a spin column filled with the above-describedion exchange resin and was sufficiently agitated. Thereafter, theblended liquid was centrifuged and spun down at 12000 G for 2 minutes.The concentration of Na⁺ and the pH of the sample were measured.

Example 2

50 mg of a strong acidic positive ion exchange resin (AG1-X8 (H⁺ type)(manufactured by Bio-Rad Laboratories, Inc.)) and 50 mg of a strongbasic negative ion exchange resin (AG50W-X8 (OH⁻ type) (manufactured byBio-Rad Laboratories, Inc.)) were used instead of 100 g of the strongacidic positive ion exchange resin (Nuvia S (Na⁺ type) (manufactured byBio-Rad Laboratories, Inc.)), 50 mg of the strong acidic positive ionexchange resin (AG1-X8 (H⁺ type) (manufactured by Bio-Rad Laboratories,Inc.)) and 50 mg of the strong basic negative ion exchange resin(AG50W-X8 (OH⁻ type) (manufactured by Bio-Rad Laboratories, Inc.)) inExample 1 and were evaluated similar to Example 1.

Example 3

70 mg of a strong acidic positive ion exchange resin (AG1-X8 (H⁺ type)(manufactured by Bio-Rad Laboratories, Inc.)) and 50 mg of a strongbasic negative ion exchange resin (AG50W-X8 (OH⁻ type) (manufactured byBio-Rad Laboratories, Inc.)) were used instead of 50 g of the strongacidic positive ion exchange resin (AG1-X8 (H⁺ type) (manufactured byBio-Rad Laboratories, Inc.)) and 50 mg of the strong basic negative ionexchange resin (AG50W-X8 (OH⁻ type) (manufactured by Bio-RadLaboratories, Inc.)) in Example 2 and were evaluated similar to Example2.

Comparative Example 1

Only 100 mg of a strong acidic positive ion exchange resin (Nuvia S (Na⁺type) (manufactured by Bio-Rad Laboratories, Inc.)) was used instead of100 mg of the strong acidic positive ion exchange resin (Nuvia S (Na⁺type) (manufactured by Bio-Rad Laboratories, Inc.)), 50 mg of the strongacidic positive ion exchange resin (AG1-X8 (H⁺ type) (manufactured byBio-Rad Laboratories, Inc.)) and 50 mg of the strong basic negative ionexchange resin (AG50W-X8 (OH⁻ type) (manufactured by Bio-RadLaboratories, Inc.)) in Example 1 and was evaluated similar to Example1.

Comparative Example 2

Only 50 mg of a strong acidic positive ion exchange resin (AG1-X8 (H⁺type) (manufactured by Bio-Rad Laboratories, Inc.)) was used instead of100 mg of the strong acidic positive ion exchange resin (Nuvia S (Na⁺type) (manufactured by Bio-Rad Laboratories, Inc.)), 50 mg of the strongacidic positive ion exchange resin (AG1-X8 (H⁺ type) (manufactured byBio-Rad Laboratories, Inc.)) and 50 mg of the strong basic negative ionexchange resin (AG50W-X8 (OH⁻ type) (manufactured by Bio-RadLaboratories, Inc.)) in Example 1 and was evaluated similar to Example1.

A summary of the evaluation results in Examples 1 to 3 and ComparativeExamples 1 and 2 are shown in Table 3.

TABLE 3 Before adsorption After adsorption treatment treatmentComparative Comparative — Example 1 Example 2 Example 3 Example 1Example 2 Ion — Nuvia S + AG mix AG mix Nuvia S AG1-X8 (H⁺) exchange AGmix resin Na⁺ 0.45 0.002 0.004 0.006 0.45 0.017 pH 4.9 5.5 5.2 5.6 5.21.9

In each Example 1, Example 2 and Example 3, the ion exchange resinincluding the H⁺ type strong acidic positive ion exchange resin and theOH⁻ type strong basic negative ion exchange resin was used, whereby eachsample was demineralized with higher accuracy as compared with those inComparative Example 1 and Comparative Example 2. Also, in Example 1,Example 2 and Example 3, the variation in the pH in the sample wasinhibited with higher accuracy as compared with Comparative Example 2.

As shown in Example 2 and Example 3, it suggested that ademineralization effect was provided and the variation in the pH wasinhibited even when the amount of the strong acidic positive ionexchange resin and the amount of the strong basic negative ion exchangeresin were the same.

5. Effect of Demineralization on LAMP Reaction and RT-LAMP Reaction

Hereinbelow, an effect of demineralization on a LAMP reaction and aRT-LAMP reaction was reviewed in Examples 4 and 5 and ComparativeExamples 3 to 5.

Example 4

100 mg of a positive ion exchange resin (Nuvia S Na⁺ type (manufacturedby Bio-Rad Laboratories, Inc.)), 50 mg of a strong acidic positive ionexchange resin (Nuvia S (Na⁺ type) (manufactured by Bio-RadLaboratories, Inc.)) and 50 mg of a strong basic negative ion exchangeresin (AG50W-X8 (OH⁻ type) (manufactured by Bio-Rad Laboratories, Inc.))were weighed and charged into a spin filter column (Ultrafree-MC, 0.45μm). Next, 50 mM MES buffer (pH of 5) was prepared. To the buffer, 1000copy/μL of bifidobacteria ultrasonically disintegrated was added, then0.9 mg/mL of NaCl was added. After a sample obtained was sufficientlyagitated at normal temperature, 200 μL of each sample was dropped into aspin column filled with a Na⁺ type or a H⁺ type strong acidic positiveion exchange resin and was sufficiently agitated. Thereafter, theblended liquid was centrifuged and spun down at 12000 G for 2 minutes.The LAMP reaction and the RT-LAMP reaction were measured to similar tothe text example 1.

Example 5

50 mg of a strong acidic positive ion exchange resin (AG1-X8 (H⁺ type)(manufactured by Bio-Rad Laboratories, Inc.)) and 50 mg of a strongbasic negative ion exchange resin (AG50W-X8 (OH⁻ type) (manufactured byBio-Rad Laboratories, Inc.)) were used instead of 100 g of the strongacidic positive ion exchange resin (Nuvia S (Na⁺ type) (manufactured byBio-Rad Laboratories, Inc.)), 100 g of the strong acidic positive ionexchange resin (AG1-X8 (H⁺ type) (manufactured by Bio-Rad Laboratories,Inc.)) and 50 mg of the strong basic negative ion exchange resin(AG50W-X8 (OH⁻ type) (manufactured by Bio-Rad Laboratories, Inc.)) inExample 4 and were evaluated similar to Example 4.

Comparative Example 3

A sample to which no NaCl was added was used and the sample was notdropped into the spin column filled with the ion exchange resindissimilar to Example 4, but was evaluated similar to Example 4.

Comparative Example 4

A sample was not dropped into the spin column filled with the ionexchange resin dissimilar to Example 4, but was evaluated similar toExample 4.

Comparative Example 5

100 mg of a strong acidic positive ion exchange resin (Nuvia S (Na⁺type) (manufactured by Bio-Rad Laboratories, Inc.)) was used instead of100 g of the strong acidic positive ion exchange resin (Nuvia S (Na⁺type) (manufactured by Bio-Rad Laboratories, Inc.)), 50 mg of the strongacidic positive ion exchange resin (AG1-X8 (H⁺ type) (manufactured byBio-Rad Laboratories, Inc.)) and 50 mg of the strong basic negative ionexchange resin (AG50W-X8 (OH⁻ type) (manufactured by Bio-RadLaboratories, Inc.)) in Example 4 and were evaluated similar to Example4.

A summary of the evaluation results in Examples 4 and 5 and ComparativeExamples 3 to 5 are shown in FIG. 8 and FIG. 9. Example 4, Example 5 andComparative Example 4 revealed that when the sample was demineralized bythe ion exchange resin, Tt values of the LAMP reaction and the RT-LAMPreaction were decreased. Example 4, Example 5 and Comparative Example 4suggest that when the sample is demineralized by the ion exchange resin,the LAMP reaction can proceed successfully. Example 4 and Example 5revealed that an effect of using the strong acidic positive ion exchangeresin (Nuvia S Na⁺ type (manufactured by Bio-Rad Laboratories, Inc.)) asthe ion exchange resin on the LAMP reaction was small. It iscontemplated that an effect of using the strong acidic positive ionexchange resin (Nuvia S Na⁺ type (manufactured by Bio-Rad Laboratories,Inc.)) as the ion exchange resin on the demineralization of the sampleis small. In addition, the LAMP reaction proceeded unsuccessfully inComparative Example 4 and Comparative Example 5. This suggests that thesample is sufficiently demineralized in order to proceed the LAMPreaction successfully.

6. Review about Additives

Hereinbelow, an effect of additives (Brij35 and EDTA) was reviewed inExample 6 and Example 7.

Example 6

100 mg of a strong acidic positive ion exchange resin (Nuvia S (Na⁺type) (manufactured by Bio-Rad Laboratories, Inc.)), 50 mg of a strongacidic positive ion exchange resin (AG1-X8 (H⁺ type) (manufactured byBio-Rad Laboratories, Inc.)) and 50 mg of a strong basic negative ionexchange resin (AG50W-X8 (OH⁻ type) (manufactured by Bio-RadLaboratories, Inc.)) were weighed and charged into a spin filter column(Ultrafree-MC, 0.45 μm). Next, 50 mM MES buffer (pH of 5) was prepared.To the buffer, 10% by volume of bovine whole blood was added, 1000copy/μL of bifidobacteria ultrasonically disintegrated was added, 0.5%by volume of Briji35 was added, and 1 mL of EDTA was added. The LAMPreaction and the RT-LAMP reaction were carried out and evaluated on theresultant sample containing nucleic acids similar to Example 4.

Example 7

A Brij35 and EDTA were not added to the sample dissimilar to Example 6,but the sample was evaluated similar to Example 6.

Results of the evaluation in Example 6 and Example 7 are shown in FIGS.10 and 11. A summary of the evaluation results are shown in Table 4.

TABLE 4 Example 6 Example 7 LAMP RT-LAMP LAMP RT-LAMP Tt (min) Tt (min)Tt (min) Tt (min) Before 23.0 12.4 19.2 9.9 adsorption treatment After19.3 10.8 28.8 12.8 adsorption treatment

In Example 6, it revealed that when the foreign substances in the samplewere adsorbed by the ion exchange resin, the Tt values in both of theLAMP reaction and the RT-LAMP reaction were decreased as compared withthose before the adsorption treatment. On the other hand, in Example 7,it revealed that when the foreign substances in the sample were adsorbedby the ion exchange resin, the Tt values in both of the LAMP reactionand the RT-LAMP reaction were increased as compared with those beforethe adsorption treatment. This may because the recovery rate of thenucleic acids was increased by adding Briji 35 and 1 mL of EDTA to thesample in Example 6 dissimilar to Example 7.

7. Review about Surfactant/Hydrophilic Polymer

Hereinbelow, an effect of a surfactant/a hydrophilic polymer wasreviewed in Examples 8 to 11.

Example 8

100 mg of a strong acidic positive ion exchange resin (Nuvia S (Na⁺type) (manufactured by Bio-Rad Laboratories, Inc.)), 50 mg of a strongacidic positive ion exchange resin (AG1-X8 (H⁺ type) (manufactured byBio-Rad Laboratories, Inc.)) and 50 mg of a strong basic negative ionexchange resin (AG50W-X8 (OH⁻ type) (manufactured by Bio-RadLaboratories, Inc.)) were weighed and charged into a spin filter column(Ultrafree-MC, 0.45 μm). Next, 50 mM MES buffer (pH of 5) was prepared.To the buffer, 10% by volume of bovine whole blood was added, 1000copy/μL of bifidobacteria ultrasonically disintegrated was added, and0.5% by volume of Briji35 was further added. In Example 7, a nonionicsurfactant or a nonionic hydrophilic polymer was not added to thesample. The recovery rate of the nucleic acids was evaluated on theresultant sample containing nucleic acids similar to Test Example 1.

Example 9

0.5% by volume of Brij35 was added as the nonionic surfactant to thesample before the adsorption treatment by the ion exchange resindissimilar to Example 8, but the sample was evaluated similar to Example8.

Example 10

Tween 20 was used instead of Brij35 as the nonionic surfactantdissimilar to Example 9, but the sample was evaluated similar to Example9.

Example 11

PEG20000 was used instead of Brij35 as the nonionic surfactantdissimilar to Example 9, but the sample was evaluated similar to Example9.

Results of the evaluation in Examples 8 to 11 are shown in Table 5.

TABLE 5 Example 8 Example 9 Example 10 Example 11 Recovery rate 56 78 7762 of nucleic acids (%)

The recovery rates of the nucleic acids in Examples 8 to 10 were higherthan that in Example 7. It exemplified that the recovery rate of thenucleic acids was increased by adding the non-ionic surfactant (Brij35,Tween20) or the nonionic hydrophilic polymer (PEG20000) to the sample tocarry out the adsorption treatment of the sample.

8. Comparison of Adsorption Treatment According to Present Technologywith Adsorption Treatment by Zeolite

Example 12 and Comparative Example 6 were carried out to compare theadsorption treatment according to the present technology and theadsorption treatment by zeolite.

Example 12

A sample was prepared similar to Example 7. The LAMP reaction and theRT-LAMP reaction were carried out and evaluated three times on threesamples each having the same composition similar to Example 7.

Comparative Example 6

50 mM MES buffer (pH of 5) was prepared. To the buffer, 5% by volume ofbovine whole blood was added, and 1000 copy/μL of bifidobacteriaultrasonically disintegrated was added to prepare a sample. Next, analkali and heat treatment was carried out on the sample, and the foreignsubstances were adsorbed by zeolite. The LAMP reaction and the RT-LAMPreaction were carried out and evaluated three times on three sampleseach having the same composition similar to Example 4.

Results of the evaluation in Example 12 and Comparative Example 6 areshown in FIG. 12 (the LAMP reaction) and in FIG. 13 (the RT-LAMPreaction). Example 12 and Comparative Example 6 revealed that thenuclear acid amplification reaction proceeded more successfully when theforeign substances in the sample were adsorbed by the ion exchange resinas compared when the foreign substances in the sample were adsorbed bythe zeolite.

INDUSTRIAL APPLICABILITY

According to the method of purifying nucleic acids according to thepresent technology, the operation is simple and the nucleic acids can beextracted in a short time with high efficiency. Accordingly, the methodcan be applied to a nucleic acid purifying treatment for the nuclearacid amplification reaction such as PCR (Polymerase Chain Reaction) andLAMP (Loop-Mediated Isothermal Amplification) and can be used to purifyonly a minor amount or an extremely low concentration of the nucleicacids included in the sample.

DESCRIPTION OF REFERENCE NUMERALS

-   1, 101 kit for purifying nucleic acids-   3 nucleic acid purifying instrument-   10 ion exchange resin-   20 first positive ion exchange resin-   30 negative ion exchange resin-   40 second positive ion exchange resin

The invention claimed is:
 1. A method of purifying nucleic acidscomprising the step of: adsorbing substances in a sample containingnucleic acids with an ion exchange resin including a positive ionexchange resin and a negative ion exchange resin, wherein the positiveion exchange resin includes a first positive ion exchange resin and asecond positive ion exchange resin having an exclusion limit molecularweight lower than that of the first positive ion exchange resin.
 2. Themethod of purifying nucleic acids according to claim 1, wherein thesubstances are adsorbed by the first positive ion exchange resin andthen are further adsorbed by the negative ion exchange resin and thesecond positive ion exchange resin.
 3. The method of purifying nucleicacids according to claim 2, wherein the sample is flowed into a columnincluding the first positive ion exchange resin in an upper layer andthe negative ion exchange resin and the second positive ion exchangeresin in a lower layer from an upper layer side.
 4. The method ofpurifying nucleic acids according to claim 3, wherein the step is foradsorbing the substances included in the sample that is diluted with abuffer solution by the ion exchange resin, and the buffer solution has apH of 4.0 to 8.0.
 5. The method of purifying nucleic acids according toclaim 4, wherein the positive ion exchange resin is a strong acidicpositive ion exchange resin.
 6. The method of purifying nucleic acidsaccording to claim 5, wherein a counter ion of the first positive ionexchange resin is Na⁺.
 7. The method of purifying nucleic acidsaccording to claim 6, wherein a counter ion of the second positive ionexchange resin is H⁺.
 8. The method of purifying nucleic acids accordingto claim 7, wherein the negative ion exchange resin is a strong basicnegative ion exchange resin.
 9. The method of purifying nucleic acidsaccording to claim 8, wherein a counter ion of the negative ion exchangeresin is OH⁻.
 10. The method of purifying nucleic acids according toclaim 9, wherein a percentage of an ion exchange capacity of thenegative ion exchange resin to the second positive ion exchange resin is50% to 150%.
 11. The method of purifying nucleic acids according toclaim 10, wherein a nonionic surfactant and/or a nonionic hydrophilicpolymer compound is used to adsorb the substances.
 12. The method ofpurifying nucleic acids according to claim 11, wherein the substancesare foreign substances containing at least a protein and a metal salt.13. A method of extracting nucleic acids, comprising the steps of:ultrasonically treating the sample including nucleic acids, adsorbingsubstances included in the sample with a positive ion exchange resin anda negative ion exchange resin, and concentrating the nucleic acids byblocking the nucleic acids migrated by electrophoresis.
 14. The methodof extracting nucleic acids according to claim 13, wherein theelectrophoresis is carried out on the nucleic acids into which anintercalator having an anionic functional group is inserted.
 15. Themethod of extracting nucleic acids according to claim 14, wherein theelectrophoresis is carried out on the nucleic acids by mixing thesample, a compound having a functional group that is reacted with acarboxyl group of the substances included in the sample by dehydrationcondensation, and a condensation agent of the dehydration condensationreaction.
 16. A kit for purifying nucleic acids comprising: a positiveion exchange resin; a negative ion resin; and a nucleic acid purifyinginstrument internally holding the positive ion exchange resin and thenegative ion exchange resin for distributing a sample including nucleicacids, wherein the positive ion exchange resin includes a first positiveion exchange resin and a second positive ion exchange resin having anexclusion limit molecular weight lower than that of the first positiveion exchange resin.
 17. The kit of claim 16, wherein the positive ionexchange resin is a strong acidic positive ion exchange resin.
 18. Thekit of claim 17, wherein a counter ion of the first positive ionexchange resin is Na⁺.
 19. The kit of claim 18, wherein a counter ion ofthe second positive ion exchange resin is H⁺.
 20. The kit of claim 16,wherein a percentage of an ion exchange capacity of the negative ionexchange resin to the second positive ion exchange resin is 50% to 150%.